贵州医科大学学报

目的 分析人脂肪间充质干细胞外泌体(hADSC-Exo)激活转化生长因子-β1(TGF-β1)/Smad同源物3重组蛋白(Smad3)-血管内皮生长因子(VEGF)信号通路促进大鼠全层皮肤缺损创面愈合的机制。方法 选取50只SPF级SD大鼠，10只提取脂肪间充质干细胞外泌体，10只作为对照组，另外30只构建全层皮肤缺损模型，共有27只大鼠建模成功，随机均分为模型组、hADSC-Exo组、hADSC-Exo+TGF-β1抑制剂组，每组9只；使用流动纳米颗粒追踪分析(NTA)和透射电镜(TEM)鉴定外泌体表征，HE染色观察病理组织、创面愈合率，ELISA检测血管生成相关因子表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)和炎症因子肿瘤坏死因子-α(TNF-ɑ)及转化生长因子-β1(TGF-β1),RT-PCR、Western blot法检测TGF-β1/Smad3-VEGF通路相关mRNA和蛋白表达量。结果 hADSC-Exo平均尺寸为118.9 nm, TEM证实其圆形纳米特征；与模型组相比，hADSC-Exo组创面愈合率及EGF、bFGF、TGF-β1水平升高，TNF-α降低(P<0.05),与hADSC-Exo组相比，hADSC-Exo+TGF-β1抑制剂组创面愈合率及EGF、bFGF、TGF-β1水平降低，TNF-α升高(P<0.05);与对照组相比，模型组TGF-β1、Smad3、VEGF mRNA与蛋白表达降低(P<0.05),经hADSC-Exo作用后表达升高(P<0.05),但在hADSC-Exo处理的同时给予TGF-β1抑制剂后升高幅度降低(P<0.05)。结论 hADSC-Exo可加快创面愈合速度的机制可能与激活TGF-β1/Smad3-VEGF信号通路、降低大鼠体内炎症反应及促使生长因子的表达有关。

Objective To analyze the mechanism of human adipose-derived mesenchymal stem cell exosomes(hADSC-Exo) for promoting wound healing in full-thickness skin defects in rats through activation of the TGF-β1/Smad3-VEGF signaling pathway. Methods Fifty SPF-grade SD rats were selected. Exosomes were extracted from adipose-derived mesenchymal stem cells of 10 rats. Another 10 rats served as the control group, and the remaining 30 rats were used to construct a full-thickness skin defect model. A total of 27 rats were successfully modeled and randomly divided into the model group, the hADSC-Exo group, and the hADSC-Exo + TGF-β1 inhibitor group, with 9 rats in each group. Exosome characterization was performed by using nanoparticle tracking analysis(NTA) and transmission electron microscope(TEM). Pathological tissue and wound healing rates were observed using hematoxylin eosin(HE) staining. Angiogenesis-related factors including epidermal growth factor(EGF) and basic fibroblast growth factor(bFGF), inflammatory factor tumor necrosis factor-α(TNF-α), and transforming growth factor-β1(TGF-β1) were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA and protein expression levels related to the TGF-β1/Smad3-VEGF pathway were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. Results The average size of hADSC-Exo was 118.9 nm, and TEM confirmed their round nano characteristics. Compared with the model group, the wound healing rate and levels of EGF, bFGF, and TGF-β1 were increased in the hADSC-Exo group, while TNF-α was decreased(P<0.05). Compared with the hADSC-Exo group, the wound healing rate and levels of EGF, bFGF, and TGF-β1 were decreased in the hADSC-Exo + TGF-β1 inhibitor group, while TNF-α was increased(P<0.05). Compared with the control group, the mRNA and protein expression of TGF-β1, Smad3, and VEGF were decreased in the model group(P<0.05), but after treatment with hADSC-Exo, their expression increased(P<0.05), while this increase was attenuated upon addition of the TGF-β1 inhibitor(P<0.05). Conclusion The hADSC-Exo can reduce the inflammatory response, promote the expression of growth factors, and accelerate wound healing in rats by activating the TGF-β1/Smad3-VEGF signaling pathway.